Lactic acid bacteria and their use in the treatment of cancer

ABSTRACT

The invention concerns the isolation of novel properties of lactic acid bacteria stains. Said novel properties are advantageously useful for preventing and treating cancer. More particularly, the invention concerns the use of lactic acid bacteria to facilitate induction of cell apoptosis of a cancer. The invention also concerns the use of lactic acid bacterial strains, such as  Lactobacillus acidophilus  and  Lactobacillus casei  in methods and compositions for preventing and treating cancer, in particular colon cancer.

INCORPORATION OF SEQUENCE LISTING

Two paper copies of the sequence listing (Sequence Listing No. 1 andSequence Listing No. 2) and a computer-readable form of the sequencelisting on 3.5 floppy disk containing the file named SequenceListing.txt, as modified on Apr. 6, 2005, are herein incorporated byreference.

AREA OF INVENTION

The present invention concerns highlighting the utility of lacticbacteria strain in the prevention and treatment of cancer. Morespecifically, the present invention concerns the use of lactic acidbacteria to facilitate the induction of cellular apoptosis of a cancer.

DESCRIPTION OF PRIOR ART

Lactobacillus acidophilus I-1492 bacteria as present in theBio-K+product and subject of patent request PCT/CA97/00915 arerecognised to have a beneficial effect on blood cholesterol levels inmammals.

The international application no. WO 98/23727 has as an object a lacticferment which comprises a strain of Lactobacillus acidophilus I-1492. Assuch, these bacteria are mainly used for the preparation of lacticferment in order to reduce blood cholesterol levels in mammals.

In addition, these bacteria are also known to possess properties thathave the effect of strengthening the immune system, facilitatingnutrient absorption and stimulating the intestinal flora. It is knownthat lactic acid bacteria have a positive effect on the intestinal floraand also on the immune system. Indeed, lactic acid bacteria allowstimulation of the immune system which thus has the effect of providinga better defense at the level of the digestive system. These bacteriaare also known to neutralize secondary effects caused by variousantibiotics.

In the country, a good number of people die every year of colon cancer.Cancer is the third disease that causes the most deaths per year. InCanada, in the year 2000, the morality rate attributed to cancer was6,500 and there are more than 17,000 new cases.

The use of neutraceuticals involving the administration of yogurt and/orof fermented milk as a complementary treatment against cancer is alsoknown.

The treatment which is already used as therapy in humans is the ablationof the cancerous mass by surgery and then there may be irradiation ofthe area where the cancer was found in order to avoid leaving traces ofunwanted cells.

Chemotherapy also exists. This treatment involves the administration ofanticancer agents such as 5-fluoro-uracil (5FU). This compound iscombined with an adjuvant to limit the negative effects of chemotherapy.5-fluoro-uracil is a medicament that is commonly used to treat coloncancer. This medicament may be administered orally or intra peritoneally(as close as possible to the cancerous target) given its largeinstability in serum. It is known for causing death in cancerous coloncells by different means.

The death of a cell, also named apoptosis, may be executed with the useof different proteins. For example, cellular apoptosis can result fromthe activation of a membrane receptor or from the cytoplasmic expressionof different proteins favoring this phenomenon. On this subject, it isknown that 5FU increases the expression of p53 protein as well as theexpression of the Fas membrane receptor.

1 Generalities on Apoptosis

1.1 Definition of Apoptosis

Discovered and rediscovered many times by different cytologists andbiologists, programmed cell death has acquired different namesthroughout the last two centuries. In 1972, the term apoptosis wasfinally adopted and invented by Currie and his colleagues in order todescribe a frequent model of programmed cell death which the authors hadobserved repeatedly in several tissues and cell types. It was observedthat dying cells shared many morphological characteristics which aredifferent from the characteristics observed in cells afflicted by apathology and in necrotic cells; and the authors suggest that theseshared morphological characteristics could be the result of a common andpreserved cell death program.

1.2 Role of Apoptosis

Researchers have discovered that the cells of our body can killthemselves, they know that this cellular “suicide”, called “apoptosis”,is essential to the organism. Apoptosis is as fundamental for thephysiology of cells and tissues as cellular division and differentiationare. Apoptosis is the most common form of physiological cell death whichoccurs at different time, for example, during embryonic development,during tissue reorganisation, during immunological regulation and duringtumoral regression. Hence, physiological cell death is a spontaneousprocess of cellular elimination, allowing to ensure cellular renewal,and which intervenes in the maintenance of cellular and tissuehomeostasis in a manner opposite to mitosis. It is the innate mechanismby which the organism eliminates unwanted cells. Each cell containswithin itself the genetic mechanism of its own destruction. The cellremains alive only at the condition that it receives the survivalsignals emitted by its environment. If the cell perceives signals whichinstruct it to commit suicide, it thus engages the death program. Amalfunction at the level of the equilibrium between the proteinsmaintaining the cell alive and the proteins leading to cell death can beassociated with a large spectrum of diseases including cancer,neurodegeneration, autoimmune diseases, diabetes, and other disorders.

2 Morphological Characteristics of Apoptosis and Necrosis

2.1 Apoptosis

One of the key characteristics of apoptosis is cellular shrinking. Whilecellular shrinking occurs, the cytoplasm compresses itself and thenuclear chromatin condenses and forms aggregates in the nucleus, whichthen stick against the nuclear membrane. The cellular organelles, suchas the mitochondrion, on the other hand, appear relatively unchanged.Afterwards, the nucleus becomes fragmented. The formation and theemission of buds are observed at the surface of the cell. The integrityof the plasma membrane, even if the permeability increases, isnonetheless preserved all along the process. During the final stage ofapoptosis, the cell breaks up into many vesicles containing a variety ofintact organelles and nuclear fragments. The apoptotic cellularfragments are rapidly engulfed by surrounding phagocytic cells such asthe macrophage. Apoptosis constitutes a so-called “clean” death sincecellular fragments are rapidly eliminated. There is no inflammatoryphase, nor lesion of the surrounding tissue and this in part becausetheir cellular membrane remains intact. In summary, the morphologicalchanges characteristic of apoptosis are cytoplasmic shrinking, DNAcondensation and fragmentation and finally the formation of apoptoticbody containing nuclear fragments surrounded by the cytoplasm and thecellular membrane.

2.2 Necrosis

Necrosis refers to a sudden death taking place after an extreme physicalor chemical stress. It is marked by different morphological criteria.During necrosis, this uncontrolled cell death, there is a rapid loss ofcontrol of the ionic flux leading to the penetration of water and anincrease in ionic influx, the cells expand as well as its organelleslike the mitochondrion and the endoplasmic reticulum until the burstingof the membranes and the non-specific fragmentation of DNA into thenucleus. The release of the cytoplasmic contents to the outside inaddition to other events, most often provokes lesions in the tissueslocated in proximity and induces a very pronounced local inflammatoryresponse.

TABLE 1 Main differences between necrosis and apoptosis CharacteristicsNecrosis Apoptosis Tissue distribution Cellular grouping Isolated cellsTissue reaction Lysis and cellular content Phagocytosis of apoptoticrelease leading to bodies by macrophages surrounding tissue or bysurrounding cells inflammation and absence of inflammation CellMorphology Expansion Shrinking, loss of contact with surrounding cells,“blebbing”, apoptotic body formation Organelles Damaged Intact NucleusDisintegrated Condensed and fragmented Lysosomes Damaged IntactMitochondria Defective, ATP deficient, Swollen, change, may swollen anddamaged break, cytochrome c release Biochemistry Non specificdegradation Intranuclear cleavage DNA Proteins Non specific degradationCaspase activation3 Different Stages of the Apoptotic Process

Apoptosis can have three different stages (FIG. 2). First the cell mustreceive an apoptotic signal therefore the activation or the commitmentphase. Then, there is a regulation or a control phase; and finally anexecution phase during which the intracellular enzymatic cascadeinducing apoptotic cell death takes place.

3.1 Apoptosis Activation Stage

A variety of stimuli, internal as well as external, can activate thecell to become apoptotic. Among the different stimuli, one can enumeratebiological agents (membrane receptors, transcription factors,oncoproteins, viral infections, bacterial toxins, . . . ), suppressionof factors that are essential to cellular growth (cytokines, growth andnutritive factors, . . . ), genomic DNA lesions (spontaneous orprovoked), exposure to chemical substances (cancer causing agents)exposure to physical inductors (UV rays, X-rays, microwaves, heat, . . .). This phase continues through many biochemical modifications.

3.2 Apoptotic Decision or Execution Stage

Following these different stimuli, the cell receives the differentsignals and decides to become apoptotic or not. This stage involvesvarious signal transduction pathways amongst others the activation (orinactivation) of serine/threonine and tyrosine kinases and phosphatases,secondary messenger synthesis, gene modification and expression, and theactivation of specialized proteases known by the name of caspases. Thefinal decision to become apoptotic depends on many factors including theequilibrium between the pro- and anti-apoptotic proteins (the family ofBcl-2 proteins), the metabolic state of the cell and also the stage ofthe cell cycle in which the cell is. Many arguments suggest that theprogression of this second stage is controlled by the family of Bcl-2proteins, which are generally associated to the external membrane of themitochondria, of the endoplasmic reticulum, and of the nucleus. Thefamily of Bcl-2 proteins is divided into two groups of proteins, thefirst inhibit apoptosis (Bcl-2, Bcl-X_(L), Bcl-w, CED-9, . . . ) and theothers favor apoptosis (Bax, Bid, Bad, Bak, Bcl-X_(S), . . . ). Beingpro- or anti-apoptotic, they have the ability to control the ionic fluxbetween various cellular compartments, especially between themitochondria and the cytoplasm. At this same stage, there is activationof the caspases in the form of an amplification system byauto-activation by the cells themselves and between each other and alsoby the release of apoptotic activation factors like AIF(apoptosis-inducing factor) and cytochrome c from the mitochondria. Theintermembrane space of the mitochondrion comprises many proteins whichparticipate in the activation of apoptosis, like pro-caspase-2, -3, -7and -9, AIF and cytochrome c. The activation of these caspases will leadto a point of no return since, by their activation, they will cleavetheir different targets, amongst others, the proteins necessary for cellsurvival.

3.3 Apoptotic Degradation Stage

The cell is now irreversibly engaged in the cell death program thatconsists in the presentation of morphological characteristics of theapoptotic signature. Therefore, by the activation of the differentcaspases, many proteins necessary for cell survival are cleaved andbecome non-functional, like the poly(DNA-ribose) polymerase. Othercaspase targets may be activated by them, amongst others, DNases whichwill cut up the chromatin into high molecular weight fragments.

4 Proteins Involved in the Mechanisms of the Regulation of Apoptosis

4.1.1 Family of Bcl-2 Proteins

The Bcl-2 protein and this protein of the same family are importantapoptosis modulators. In this protein family, there are two classesincluding the anti-apoptotic proteins (Bcl-2, Bcl-X_(L), Mcl-1, Bcl-w,Bfl-1/A1, Brag-1) and the pro-apoptotic proteins (Bax, Bak, Bad, Bid,Bik, Bim, Bcl-X_(S)) (Reed, 1996). The members of the Bcl-2 family areclassified by the number of domains homology with Bcl-2 (BH:Bcl-2homology). The Bcl-2 protein contains 4 domains. All the anti-apoptoticproteins have the four domains, whereas the pro-apoptotic proteins canbe divided in three categories. One group contains the BH1, BH2 and BH3domains (Bax, Bad), whereas the other group contains only the BH3 domain(Bad, Bid, Bik). Bcl-Xs form_(s) its own group, containing the BH3 andBH4 domains. A crystallography study allowed to determine the structureof the Bcl-X_(L) protein. The protein is therefore formed by two centralα-helices surrounded by five amphipathic α-helices. It is interesting tonote that the tri-dimensional structure is homologous to bacterialtoxins forming pores in membranes, like the diphtheria toxin andcolicin, which may suggest a potential action mechanism of theseproteins at the mitochondrial level. Another structural characteristicis the capacity of these proteins to homo- and heterodimerize with oneanother through their BH3 domain, as such, they may favor or antagonizetheir functions between each other.

4.1.2 Roles of the Pro- and Anti-Apoptotic

A—On the mitochondrion

First of all, a description of the modifications that the mitochondriongoes through during apoptosis. The isolated mitochondria in a situationof apoptosis goes through what is known as a mitochondrial permeabletransition (MPT). Experimentally, MPT is characterized by an abruptincrease in the internal membrane permeability to particles with amolecular weight of ≦1500 Da. This transition in permeability has manyconsequences including the collapse of Δψ_(m), osmotic swelling, releaseof matrix Ca²⁺, the creation of oxygen-reactive species, and the ruptureof the external membrane of the mitochondrion leading to the release ofcytochrome c from the inter-membrane space of the mitochondrion. Abiochemical characterization of the mitochondrion has allowed toidentify Ca⁺² and voltage-sensitive pores which control MPT, which willbe named PT pores. The pores are localized at the junction of theinternal and external membranes of the mitochondrion, and as such, theopening of the pores allow direct communication between the matrix ofthe mitochondrion and its environment. The “voltage-dependent anionchannel” (VDAC) and the “adenine nucleotide translocator” (ANT) are partof the PT pores.

The proteins of the Bcl-2 family have various cytoplasmic distributions.The Bcl-2 and Bcl-x proteins, have a hydrophobic C-terminal tailcontaining a membrane insertion sequence, and the majority of theseproteins are recognized to be associated to the membranes of themitochondria, of the endoplasmic reticulum, and to the nuclear membrane.In their inactive form, the pro-apoptotic proteins, Bad, Bax and Bid,have a location which is primarily cytoplasmic. However, during theiractivation, they are relocated on the mitochondria. When Bid is cleaved,its COOH terminal is relocated to the surface of the mitochondrion. Theaction of these proteins, either to prevent or to initiate apoptosis, islocated at the level of the mitochondria, however the mechanism of theseactions remains controversial and uncertain. It has been demonstratedthat by adding the protein Bax, a pro-apoptotic protein, to isolatedmitochondria, there is induction of cytochrome c release, whereas overexpression of Bcl-2 or Bcl-X protein prevents the release of cytochromec thus blocks apoptosis. It is clear that the Bcl-2 family is implicatedvery narrowly in the liberation of cytochrome c, the electron transportprotein within the mitochondrion. In addition to its implication inoxidative phosphorylation in the mitochondrion, cytochrome c (as well asthe adaptor protein Apaf-1) is one of the components required forcaspase-9 activation in the cytosol. How can the members of the Bcl-2family regulate the release of cytochrome c? Many hypotheses have beenput forth, however none has been definitively proven. There are threebasic models that may be suggested.

1—The members of the Bcl-2 family form a channel which facilitates thetransport of proteins. Based on the structural similarity between theBcl-X_(L) and the sub-unit of the diphtheria toxin which form pores, ithas been suggested that the Bcl-2 proteins may insert themselves in theexternal membrane of the mitochondrion, where they may form a channel oreven a big hole. The members of the Bcl-2 family may indeed insertthemselves in synthetic bi-lipid layer, oligomerize, and form a channelwith a discrete conduction. The Bid and Bik proteins may directly inducethe mitochondrion to release cytochrome c without interacting with VDACor ANT suggesting that they act outside of the PT pores.2—The members of the Bcl-2 family interact with other proteins to formchannels. The family of Bcl-2 proteins interacts with many proteins. Onepossibility is that the members of the family of pro-apoptotic proteinrecruit other proteins from the external mitochondrial membrane in orderto form a pore large enough to form a channel. A particularlyinteresting candidate for such a protein would be the voltage dependentanion channel (VDAC), many members of the Bcl-2 family can bind to itand regulate its channel activity. Since the characterized size of thepore of the VDAC channel is too small to let the proteins through, thismodel must assume that the VDAC goes through a conformational changeafter binding of members of the Bcl-2 family. It has been demonstratedthat the Bcl-2 and Bcl-XL proteins favor the closing of the PT pores,whereas the pro-apoptotic protein Bax has the contrary effect; itinteracts with ANT and VDAC to favor the opening of these pores and theanchoring of cytochrome c.3—The Bcl-2 family members induce a rupture in the external membrane ofthe mitochondrion. It is possible that the Bcl-2 family controls thehomeostasis of the mitochondrion. In this model, the apoptotic signalwould alter the physiology of the mitochondrion (for example ionicexchange or oxidative phosphorylation) such as the organelle swellings,which results in the physical rupture of the external membrane and there-dumping of proteins located between the membranes of themitochondria, in the cytosol. The need to form a channel big enough tolet cytochrome c go through is now not necessary since the proteinswould simply diffuse through the tears in the double lipid layer.

The pro-apoptotic proteins (Bid) may homodimerize to form a pore to letthe cytochrome c exit. The anti-apoptotic proteins (Bcl-2) have thecapacity to bind with PT pores and thus prevent the re-dumping ofinter-membrane proteins, however, the pro-apoptotic proteins (Bax), willallow the opening of PT pores.

The AIF (for apoptosis-inducing factor) protein which has beenidentified and its gene cloned, is capable, by itself, to induceapoptosis in the isolated nuclei. This molecule is synthesized in thecytosol under a precursor form and is then imported into themitochondrion. Like cytochrome c, this is a phylogenetically ancientmolecule, with a double function; oxydoreduction and apoptogeneticfactor. However, in contrary to the pathway of cytochrome c, whichnecessitates the activation of other factors to induce apoptosis, thepathway of AIF is instead independent of caspases and does notnecessitate any intermediate in order to provoke apoptosis. Thisconstitutes amongst others a prototype of the apoptotic pathwaysindependent of caspases.

The hypothesis concerning inhibitory apoptotic mechanisms and inparticular the sequestration of Apaf-1 by Bcl-2 and its anti-apoptoticagonists seems to still be discussed. Apaf-1 is probably an importanttarget of the Bcl-2 family members, since Apaf-1 deficient cells arerefractory to different pro-apoptotic signals that are themselvesinhibited by Bcl-2. In addition, an over expression of Apaf-1 hasdemonstrated that this protein was associated with survival proteinslike Bcl-X_(L) and Bcl-2. Also it has been shown that noco-immunoprecipitation between the members of the Bcl-2 family andApaf-1 exists. Apaf-1 has also been found at the level of sites wherethe survival proteins like Bcl-2 and Bcl-X_(L) reside, like externalmitochondrial membranes, the nuclear membrane and the endoplasmicreticulum.

4.1.3 Modulation Mechanisms of the Proteins of the Bcl-2 Family

Many different mechanisms exist to modulate the functions of pro- andanti-apoptotic proteins. Firstly, the state of dimerization of membersof the Bcl-2 family affects their activity. One of the functions ofanti-apoptotic proteins Bcl-2 and Bcl-X_(L) is to dimerize with thepro-apoptotic protein Bax in order to neutralize its activity. By beinga heterodimer, Bax is inactive, but once it is free to dimerize withitself, Bax is able to induce apoptosis. Bid, Bik and Bad may act byinhibiting the apoptotic action of Bcl-2 and Bcl-X_(L) by formingheterodimers. Secondly, altering the expression level of pro- andanti-apoptotic members of the Bcl-2 family may either initiate orinhibit apoptosis. For example, when the number of Bcl-2 is larger thanor equal to the number of Bax, the cell in question is protected fromapoptosis. However, when the number of Bax exceeds the number of Bcl-2,the cell is more subject of becoming apoptotic. Thirdly, the proteins ofthe Bcl-2 family may be modified by phosphorylation. The best examplefor this would be the pro-apoptotic protein Bad. In itsnon-phosphorylated state, it dimerizes with Bcl-2 and Bcl-X_(L) therebyneutralizing its anti-apoptotic activity. On the other hand, when Bad isphosphorylated, it is sequestered and hence cannot interact andneutralize Bcl-2 and Bcl-X_(L). Fourthly, the Bcl-2 family can bemodified by cleavage. When apoptosis is caused by Fas, it has beendemonstrated that the caspases would cleave Bcl-2 and Bcl-X_(L) and thecleaved products are no longer protectors and even become pro-apoptotic.Bid is another protein of the Bcl-2 family which is activated by thecleavage of the caspases. While the protein in its entire length isinactive, following cleavage caused the by caspase 8, Bid induces there-dumping of cytochrome c by the mitochondrion. Finally, theconformation of Bcl-2 proteins modifies their activity. The bestevidence for this mechanism comes from the studies done on Bax. In itsinactive state, Bax exists in a conformation in which it resistsproteolytic cleavages. However, following its activation and itsre-localization on the mitochondrion, the N-terminal region of theprotein becomes susceptible to cleavages, suggesting that aconformational change indeed occurs.

To summarize, the mitochondria have an important place in the launchingof apoptosis. Their inter-membrane space contains many proteins(cytochrome c, caspase-2, -3, -7, and -9, AIF) which once released inthe cytoplasm participate in the degradation phase of apoptosis. Theenigma of the induction mechanisms and of the apoptosis controlmechanisms by the mitochondria rests on four essential points: themolecules of the Bcl-2/Bcl-X_(L) family which could contribute to theformation of ion channels at the level of intracellular membranes. Thepro-apoptotic members of this family (like Bax, Bid, . . . ) may alsointervene in the permeability of the PT pores of the mitochondrialmembrane, notably as apoptosis activator proteins. Lastly, theanti-apoptotic molecules of the Bcl-2 family may also act by titratingendogenous activators (like Apaf-1) of apoptosis. A last point is thatcertain pro-caspases also have a mitochondrial localization. As such,the apoptotic promotors or inhibitors of the Bcl-2 family regulateapoptosis thanks to the multiple effects on the cascades of the caspasesactivation, on the redox potential, and on the function of thepermeability barrier of the mitochondrial membranes. Overall, theseobservations thus suggest an implication of the transition of thepermeability in the regulation of apoptosis induced by the mitochondria.

4.2 Role of Caspases in Apoptosis

4.2.1 Definition and Classification of Caspases

The caspases are specialized proteases which are essential forapoptosis. They are different from other proteases because they use acysteine for catalysis and they only cleave after aspartic acidresidues. This unusual specificity of having an aspartate as a substrateis found only with another protease, the granzyme B, however this enzymeuses a serine as an active site. The caspases are synthesized as asimple chain of polypeptides and they are inactive zymogens. Thesezymogens are composed of three domains: an N-terminal pro-domain, andtwo other domains, p10 and p20, which are found in the mature enzyme.When they are activated, each polypeptide chain is cleaved into twosub-units, a large one (p20) and a small one (p10), which laterdimerize. Therefore, the mature enzymes which have been observed areheterotetramers composed of two p20/p10 heterodimers and two activesites. The N-terminal peptide is cleaved and released during activation.This N-terminal peptide is not required for enzymatic activity, its roleis known on caspase 8 and 10 where it acts as an interaction domain withother proteins to modulate their activation. Caspases 8 and 10 contain a“death-effector domain” (DED) whereas caspases 2 and 9 contain a caspaseactivation and recruitment domain (CARD).

There are at least 14 different caspases identified in mammalian tissuesto this day. It is possible to divide the caspases into three differentgroups by their substrate specificity, that is, by their recognition ofthe three amino acids which precede the aspartic acid. The first groupcontains the caspases involved in the inflammatory process, thusactivation of pro-cytokines which include caspases 1, 4 and 5. Theseenzymes are sometimes known as ICE-like caspases because another namefor caspase-1 is “interleukin-1 converting enzyme” (ICE). Thetetrapeptide motif that they recognize and prefer is WEHD, on the otherhand, the ICE-like caspases are the most tolerant for what concernsamino-acid substitution when compared to signalling and effectorcaspases. The second group of caspases comprises the caspases 6, 8, 9and 10. These enzymes are considered as signalling caspases because theymay activate other caspases and thus begin the cascade. Theirrecognition motif is (LV)EXD. The last group contains the caspases 2, 3and 7. These enzymes are known as “effector” because they cleave manycellular targets which result in the morphological appearance ofapoptosis. The activation of these caspases generally end in a “point ofno return” in cellular death. The effector enzymes are the mostspecific, with the necessity of having an aspartic acid in the first andfourth positions preceding the cleavage site. Their recognition motif isDEXD. The most recent caspases, the caspases 12-14, have not yet beencharacterized enough to be classified in one of the three groups.

4.2.2 Activation of Caspases

There are three different mechanisms to activate the caspases. The firstmechanism is the activation of a caspase by another caspase which wasactivated beforehand. Most of the caspases are activated following aproteolytic cleavage of the zymogen between the p20 and p10 domains, andusually another cleavage between the pro-domain and the p20 domain. Itis interesting to note that all of these cleavage sites occur after anaspartate, the substrate of caspases, which suggests the possibility ofan activation by auto-catalysis. Indeed, the easiest way to activate acaspase is to put it in the presence of another caspase which is alreadyactivated. This strategy of caspase cascade is largely used by the cellfor the activation of three important caspases, caspase-3, -6 and -7.These three effector caspases are considered the hardest working of thefamily of caspases, and are usually more numerous and active than theothers.

As illustrated in FIG. 4, the first cleavage occurs between the p20 andp10 domains (here 12 kDa) in order to separate the two sub-units. Thesecond proteolytic cleavage occurs between the pro-domain and the largesub-unit and then there is formation of a heterotetramer which leads tothe mature caspase in its active form.

The caspase cascade is a very useful method to amplify the pro-apoptoticsignal, but it cannot explain how the first, the most downstream of thecaspases, is activated. There are at least two other models which mayexplain the activation of the very first caspases. The first is theinduction of activation by bringing them closer. It is known thatcaspase-8 is the initiator caspase when apoptosis is induced by thedeath receptors. When the ligand binds to its receptor, the deathreceptor CD95/Fas trimerize and form signalling complexes bound to themembrane. These complexes recruit, by adaptor proteins, manypro-caspase-8 molecules which results in a large local concentration ofzymogens. This caspase activation model through bringing them closerstipulates than under this crowded condition, the weak proteolyticintrinsic activity of pro-caspase-8 is sufficient to allow mutualcleavage of pro-enzymes and to activate one another. The last caspaseactivation model is the association of the pro-caspase with a regulatingsub-unit. Take for example caspase-9 which necessitates an associationwith cofactors for its activation. The “apoptotic protease activatingfactor-1 (Apaf-1) cofactor” was identified by a biochemical approach asbeing one of the two proteins necessary for the activation of caspase-9,the other being cytochrome c. The complex formed by these threeproteins, needing ATP, gives the active form of caspase-9 often calledapoptosome. Therefore, Apaf-1 is not only an activating protein ofcaspase-9 but is a sub-unit essential to its functioning. In summary,the effector caspases are generally activated by downstream caspases,whereas the initiator caspases are activated by regulatedprotein-protein interactions.

4.2.3 The Victims of Caspases

The caspases cleave a good number of cellular proteins, and the processof proteolysis is limited since there are a small number of cuts thatare achieved. Sometimes, the cleavages lead to the activation of theprotein, and at other times, the inactivation but never the degradationsince the specificity of their substrates distinguishes the caspases asbeing the most strict endopeptidases. The caspases cleave many cellularproteins whose number does not cease to increase. Structural, nuclearand signalling proteins are the targets of the caspases (Table 1). Thereare different cytoskeletal proteins cleaved by caspases, like, forexample, laminin, α-fodrin, and actin. The cleavage of these proteins isprobably responsible for the morphological changes observed duringapoptosis, for example, the cleavage of nuclear laminins is necessaryfor nuclear shrinking and budding. DNA fragmentation is due to theactivation of the “caspase-activated DNase” (CAD) by the caspase-3. ThisDNase exists in an inactive complex form with an inhibitory sub-unit,ICAD. CAD activation occurs therefore by the cleavage of the inhibitorysub-unit by caspase-3 resulting form the release and the activation ofthe catalytic sub-unit.

TABLE 2 A few examples of the victims of caspases Category Target EffectSignalling Other caspases Activation PKC δ Activation, nuclearfragmentation Phospholipase A₂ Activation Bcl-2, Bcl-X_(L) Pro-apoptoticfragment formation Bid Activation ICAD CAD endonuclease activationNuclear DNA fragmentation factor DNA fragmentation Of Polymerase polyInactivation (ADP-ribose) DNA-dependent protein Inactivation kinase U1(70 kDa)-snRNP Reduction of RNA synthesis Laminin A and B Nuclearlaminin disassembly Structural Actin Cytoskeletal rearrangement GelsolinCytoskeletal rearrangement α-fodrin Membrane change4.3 The p53 Protein

The p53 protein is a transcription factor which plays a critical role incancer prevention. The p53 protein is considered as being the “guardianof the genome”. This protein is a good example of how the decisionbetween apoptosis or life can be made at an activated verification pointwhen DNA is damaged. Depending on the stimulus and on the phase in whichthe cell is, the activation of p53 can lead to a halt of cellularproliferation and to the repair of DNA or to apoptosis. Whereas thefirst stimulus for activating p53 is damaged DNA, other cellularstresses like metabolite privation, physical damage, heat, and oxygendeprivation may also activate p53.

The level of p53 increases drastically in the few minutes following thedamage that the cell has undergone. This increase is possible bypost-translational modifications of the p53 polypeptide, without a cleardramatic induction of p53 mRNA following the damage caused to the DNA.The modification that occurs on the p53 polypeptide after damage to theDNA is translated by phosphorylation. In a cell not having undergonestress, the p53 protein has an extremely short half-life but becomesmuch more stable following a damage caused to the cell. The instabilityof the p53 protein in normal conditions for the cell is related to thefact that p53 is the target of proteolysis induced by small peptides,ubiquitins. Therefore, p53 is found to be tagged by the ubiquitins withthe help of a protein named Mdm2, a protein that plays a role in thenegative regulation of p53. It is shown that the Mdm2 protein interactswith p53 in order to become the prefect protease target by theubiquitin. The Mdm2 protein also causes the translocation of p53 fromthe nucleus to the cytoplasm, where it will undergo ubiquitin-inducedproteolysis. Therefore, by phosphorylation of the regulatory domain inthe C-terminal portion of p53, it is shown that the activation of itsbinding to DNA according to specific sequences occurs. In addition,phosphorylation of serine-15 and serine-20, at the N-terminal of p53,causes the inhibition of the interaction between p53 and Mdm2consequently increasing the level of p53 and converting it into a formwhich is capable of transcriptional activity. A large number of kinasesphosphorylate p53, including the kinase casein, the kinases linked toextra-cellular signals, protein kinase C and the kinase Raf-1. Oncephosphorylated, p53 acts like a transcription factor to increase anddecrease the transcription of many genes involved in apoptosis.

Many regulating proteins of the cellular cycle are induced by p53, forexample p21, GADD45 and members of the 14-3-3 family. The ability of p53to induce stopping of the cellular cycle in the G₁ phase following DNAdamage is well known and may be explained by the fact that the p53protein, once stimulated, has a transcriptional activity which allowstranscription of the inhibitory gene of the cyclin-dependant kinase(Cdk), the p21 protein. An elevated number of p21 will then inhibit thecyclin kinases E/Cdk2 and cyclin A/Cdk2, thus preventing these kinasesof promoting the progression of the cellular cycle. In addition, the p53protein is also involved in stopping the cellular cycle in the G₂ phasepartly because p53 induces the expression of the protein sigma 14-3-3which will cause the sequestration of the cyclin complex B/Cdc2. The p53protein may lead to apoptosis by activation of the transcription ofdifferent genes yielding proteins involved in the apoptotic process. Theproteins that are induced are the protein Bax, the Fas receptor and DR5(receptor for the death ligand TRAIL), which are all involved in theapoptotic process. It also causes the reduction of Bcl-2 mRNAexpression, thereby favoring the process of apoptosis. There seems toexist an apoptotic pathway induced by p53 which does not requirecytochrome c release but that always requires caspase activation.Eventhough the expression of the Bax protein is increased, it is ratherlocated in the cytosol and no translocation on the mitochondrion isdetectable. Therefore, there may exist another pathway by which the p53protein would induce apoptosis without the release of cytochrome c.

The final results following DNA damage may be the stopping of thecellular cycle, thus of growth, or apoptosis. A damage to the DNA yieldsan accumulation and activation of the p53 protein (FIG. 5). Onceactivated, p53 has a transcriptional activity which will increase thetranscription of different genes (GADD45, 14-3-3, Mdm2, p21, Bax, Fas,DR5). It can also down-regulate different genes (Bcl-2). By increasingp21 which will inhibit the cyclin-dependent kinases (cdk), the cellularcycle is thus stopped in G₁. The cellular cycle may also be stopped inthe G₂ phase by the increase of the proteins GADD45 and 14-3-3 by p53.The process of apoptosis is accomplished by different proteins (Bax,Fas, DR5) that are upregulated by p53. A regulatory loop of the p53protein is possible thanks to the increase of Mdm2, a protein that bindsto p53 and favors its degradation.

4.4. Death Receptors

The death receptors are receptors located on the surface of the cell andare thus named because upon binding with their ligand, they may beginthe process of apoptosis. These receptors are part of the TNF receptorfamily, in particular the TNF-1 receptor itself, the Fas receptor (alsocalled CD95 or Apo-1) and also the DR-3, DR-4 and DR-5 receptors. Thesereceptors are activated by their ligand that is soluble ormembrane-bound like “tumor necrosis factor-α” (TNF-α), Fas-L and“TNF-related apoptosis inducing ligand” (TRAIL). The death receptorligands are part of the cytokine family TNF-α, and are homotrimericmolecules. Crystallographic analyses indicate that each ligand monomerbinds to a receptor which indicates that the binding of a ligandinvolves the trimerization of these receptors. The ligand-receptorinteraction induces the trimerization of the receptor which allows thephysical association of adaptor proteins with the interacting proteindeath domains (RIP-DD), favors recruitment and activation of proximalcaspases like pro-caspase-8, -10, and -2 then capable of transmittingthe death signal inside the cell. Take for example TNF-R1 receptoractivation. By the binding of TNF-α to the TNF-R1 receptor, the lattertrimerizes giving as a result the aggregation of death domains, allowingthe recruitment of TRADD which in turn recruits the adaptor moleculeTRAF2, “TNF receptor-associated factor 2”, which leads to the activationof the JNK and NF-κB pathways. TRADD may also recruit FADD and RIPleading to the apoptotic process and to the activation of NF-κBrespectively. RIP may also recruit RAIDD, RIP-associatedICH-1/CED-3-homologous protein with a death domain, which will thenrecruit caspase-2 and induce apoptosis. When we take the Fas receptor asa model of activation for apoptosis, the Fas complex and FADD willrecruit pro-caspase-8 which will form the complex which will induce thedeath signal, “the death-inducing signal complex (DISC)”. Onceassembled, the DISC will cause a rapid auto-activation of caspase-8which will activate caspase-3 and will cause apoptosis of the cell.Thus, this first path of action of the Fas receptor is a rapid pathwaywhich short-circuits the mitochondrion and which does not necessitatethe supply of other molecules because it is based on the interaction ofpreexisting molecules.

However, it has been shown recently that the activation of caspase-8,following Fas receptor trimerization, could also provoke the cleavage ofBid, a pro-apoptotic protein of the Bcl-2 family. This cleavage bringsabout the penetration of a trunkated form of Bid into the mitochondrionwith, as a consequence, the exit of cytochrome c and the depolarizationof the mitochondrial membrane potential and apoptosis. It is also shownthat when there is activation of DR-4 and DR-5 by TRAIL, caspase 8 isactivated. The pathway used has not yet been well elucidated but manyconfirm that there is activation of caspases-3 and -9 following theactivation of caspase-8 and observe that Bid is cleaved followingcaspase-8 activation. It is possible that by the binding of TRAIL to theDR4 or DR5 receptors there is activation of caspase-8 by recruitmenthelped by an adaptor molecule which activates caspase-3 and cleaves Bidat the same time so that the latter becomes active and allows there-dumping of cytochrome c which will have the effect of activatingcaspase-9 which in turn will cause an amplification of the caspasecascade and will give death to the cell.

In summary, there would thus be at least two apoptosis signaltransduction pathways by certain death receptors, a rapid and directone, the other slower and putting into play the mitochondrial relay.This is why certain authors classify cells (type I or type II) accordingto their mode of inducing apoptosis by Fas. Like for example, theactivation of Fas, in certain cells, leads almost exclusively to thecaspase cascade only (type I cells). These cells usually do notdemonstrate any involvement of the mitochondria, and cellular death isnot inhibited by Bcl-2 or Bcl-X_(L). In other cells, the activation ofFas engages the pathway using mitochondria for the large part, and thisfollowing activation of caspase-8. The anti-apoptotic proteins Bcl-2 orBcl-X_(L) may inhibit apoptosis only in type II cells by their action ofpreventing the release of cytochrome c into the cytosol.

Many stimuli can initiate apoptosis, however common morphological andbiochemical alterations are observed and this independently of theinitial stimulus. Studies suggest that the majority of apoptotic signalsconverge on a limited number of pathways leading to apoptosis.

FIG. 6 shows the structure of members of the death receptors and theirinteractions with the principal cytoplasmic effectors implicated in theapoptotic pathways. The red arrows indicated a direct activation ofcaspase-8, and the black arrows an inhibition of apoptosis withintermediate stages (activation of kinases/transcription factor). Theabbreviations mentioned in this figure have the following meaning: DD,death domain; TRADD, TNF-receptor associated death domain; FADD,Fas-associated death domain; DISC, death-inducing signalling complex;RIP, receptor-interacting protein; TRAF2, TNF-receptor associatedfactor-2; NF-κB, nuclear factor kappa B, I-κB, inhibitory kappa B; JNKK,JNK kinase; TNF, tumor necrosis factor; TNFR, TNF receptor; Fas L, Fasligand; TRAIL, tumor necrosis factor-related apoptosis inducing ligand;DR4-5, death receptor 4-5; DED, death-effector domain; RAIDD,RIP-associated ICH-1/CED-3-homologous protein with a death domain.

The process of apoptosis requires the participation of many pathways inorder to activate the caspases (FIG. 7). The two most well known andbest characterized pathways are apoptosis signal transduction by thedeath receptors and the other pathway more internal to the cell isapoptosis induced by the changes in mitochondrial integrity,particularly the release of apoptogenetic factors like cytochrome c andAIF. There exists interconnections between these two signalling pathwaysand signal amplification loops. Abbreviations: AIF: apoptosis-inducingfactor; tBid, trunkated protein.

4.5 Protein Kinase C and Nurr77

The kinase C proteins (PKC) are part of a family of serine/threoninekinase. There are at least 11 different isoenzymes of PKC that we candivide into three sub-groups, based on their structure and theirresponse mechanism to regulatory factors. The conventional PKC (α, βI,βII, γ) are Ca²⁺-dependent and are activated by diacylglycerol (DAG) orby 12-o-tetradecanoylphorbol-3-acetate (PMA) in an in vivo fashion. Thesecond sub-group (δ, ε, η, θ, μ), the new iso-types, does not respond toCa²⁺ but is activated by DAG and PMA. The last sub-group (λ, ξ, ι), theatypical PKC, are insensitive as much to Ca²⁺ as to DAG. The PKC areresponsible for the transduction of many cellular signals during avariety of cellular processes such as cellular growth, differentiation,malign transformation and apoptosis. The PKC are also known to modulatethe activity of many different membrane proteins like the transportproteins, the channels, and the cytoskeleton-related membrane proteins.Since they have different roles, we notice that their activation cangive different results which may even be opposite results. It has beendemonstrated that PKC activation induces apoptosis in a line of gastriccancer cells treated with PMA and this through the intermediate ofcaspase-3 and serine proteases. It has also been demonstrated that PKCinhibits Fas receptor induced apoptosis and by the modulation ofpotassium (K⁺) loss and the inhibition of caspase-8 and -3 activity. Itis demonstrated by different researchers that the PKC have a role intranscriptional regulation of Fas and FasL gene expression. Park's teamhas demonstrated that the ability of PKC to induce Fas expression ispossible thanks to the TDAG51 gene (T-cell death-associated gene) inwild type cells only. There exists another mediator in the Fas/FasLexpression system that is a member of the orphan nuclear steroidreceptor superfamily Nurr77. Nurr77 plays a role in cellular growth byits role as a nuclear transcription factor. It has been shown that byadding Nurr77 in its transgenic form to thymocytes, an increase of Fasligand is obtained which suggests that Nurr77 may lead cells to becomeapoptotic by the induction of Fas ligand expression. Another role wasidentified for Nurr77. Nurr77 would be able to regulate apoptosis by ameans independent of its transcriptional regulation activity, amongother things by its re-localization from the nucleus towards themitochondria causing the release of cytochrome c. Therefore, by its roleas a transcription factor and its role as a protein causing the releaseof cytochrome c, Nurr77 can thus bring a cell to become apoptotic.

5 Colon Cancer and Defence Mechanisms

Within a tissue, homeostasis is maintained by the balance betweencellular growth and programmed cell death. A cancerous cell may bedefined as a cell which has survived the apoptotic process and maybelong to the portion of cells that may contribute to tumor formation.Many causes can lead to cancer, as much a flaw in the growth process asa flaw in the apoptotic process. These flaws are responsible for manydiseases including cancer. An accumulation of cells may occur when thedeath rate is normal but the growth rate is abnormally high or when thegrowth rate is normal but the death rate is abnormally low. Malignantcellular transformation and tumoral progression are complex processeswhich necessitate many genetic alterations.

Normally, tumoral cells are eliminated by presentation, at the membranesurface of antigens to cytotoxic T lymphocyte (CTL) and to “naturalkiller” (NK) of major histocompatibility complex class I (MHC I) attheir surface thereby allowing the activation of the immune response.The cytotoxic cells can recognize tumoral cells by the expression, ontheir surface, of viral antigens (non-self, neo-antigens (issued frommutated self-proteins), non-mutated but over-expressed self antigens,oncofeotal antigens (gene silenced during embryogenesis which would bespontaneously re-transcribed).

Sometimes, the tumoral cells develop alternatives in order to evade theimmune response. Various mechanisms are created by the tumoral cell,some of which cause an aberration in cancer cells surface antigenpresentation. The reduction of MHC I expression (the tumoral cell is nolonger recognized by the CTL, but may be destroyed by the NK cells) andthe alteration of the structure of MHC I a bad interaction between thecytotoxic cells and the tumor cells: the reduction of the secretion ofco-stimulatory or adhesion molecules (which are essential in antigenicpresentation, and may cause anergy), the reduction or mutation of themembrane Fas receptor and of receptors DR4 or DR5 (tumor cell lesssensitive to the attack of CTL or NK). The tumor cells may also secretecytokines which enhance their growth. A study demonstrates that incertain types of colon cancer, the production of IL-10 (which has aninhibitory effect on the production of CD4+ T cells of type Th1 and onmacrophage functions) is regulated by the local production ofpro-inflammatory cytokines such as IL-6 and IFN-γ. A second study on 9colon cancer lines demonstrates that these tumor cells produceimmunosuppressant factors inhibiting the proliferation of T cells.

The concept of cellular immunity is based on the capacity of cytotoxic Tlymphocytes to eliminate tumor cells. CTLs act by inducing apoptosis incancerous cells by way of two mechanisms: by the death receptors and bythe perforin/granzyme B path. However, in certain cases, the tumor cellsdevelop means to counter-attack the surveillance of the immune systemand ensure that the apoptotic process is not initiated. Teams ofresearchers have demonstrated that cancerous colon cells can defendthemselves against CTLs by surface membrane expression of Fas ligand.This causes the death of CTLs by the binding of Fas receptor on the CTLand Fas ligand of cancerous cells. On the other hand, contradictoryresults have refuted this hypothesis. Therefore, we can see that thishypothesis remains a controversial research topic. Another mechanism maybe developed by cancerous cells in order to avoid apoptosis andneutralize CTLs: secretion of Fas ligand in soluble form. Therefore, inthis scenario, the cancer cell secretes soluble Fas ligand while causinga mutation in the trans-membrane domain of the protein, thereby removingits anchoring point to the cellular membrane. Thus, the Fas ligand bindsto its ligand situated on the surface of the CTL and induces apoptosisof the CTL. It has been demonstrated that in certain cases, CTLs mayinduce apoptosis in cancerous cells which express Fas ligand by theperforin/granzyme B mechanism. On the other hand, in other cases, thetumor cells have developed a mechanism to counter-act theperforin/granzyme B pathway's efficacy by the over-expression of theserine-protease inhibitor PI-9.

The tumoral cells may develop many mechanisms to avoid death butsometimes these mechanisms can cause the cell to commit suicide or tocause fratricidal death (the death of a neighboring cancer cell). Forexample, if the cell secretes Fas ligand at its surface or in a solubleform, it may bind to Fas receptors of neighboring cell or even to a Fasreceptor located on the surface of the same cell.

There exists other ways for a cancerous cell to escape apoptosis, amongother by the mutation of certain proteins involved in the apoptoticprocess like for instance the p53 protein. This protein is one of themost common targets of colon cancer. Thus, by mutation which deprives itfrom its functions as a guardian of the genome and of activating theapoptotic pathway; even if the cell is damaged at the DNA level bydifferent treatments (radiotherapy and/or chemotherapy) it may stillescape apoptosis. There is also the over-expression of the proteinc-FLIP, which inhibits the binding between recruiting FADD proteins andbetween caspase-8 and FADD. It has been demonstrated that theover-expression of this protein is frequent in different types of coloncancer which may contribute to tumor transformation in vivo.

Naturally, the list of evasion mechanisms may be long because cancercells seem to develop strategies permitting them to overcome manyobstacles which we do not cease to discover.

5.1 Therapies Against Colon Cancer

The treatments against colon cancer have a high success rate when it islocalized in the colon and it has not gone through the walls of thecolon. We attribute this high success rate to diagnosis early in thedevelopment of the disease. According to Statistics Canada, colon canceris the third cancer causing the most deaths in Canada per year, 6,500deaths in the year 2000 and 17,000 new cases. Many treatments are nowavailable in order to counter this disease.

5.1.1 Duke's Stages of Colon Cancer

The treatments are given according to the stage of the disease. Thereare two systems to classify at which stage a patient's colon cancer is:the Duke classification and the TNM (tumor characteristics, nodalinvolvement and amount of metastasis) system. The Duke classification isthe most used and here is a summary: the A stage represents the phasewhere the colon cancer is limited to the mucous or sub-mucous membraneof the colon. Treatment options at this stage are either a colectomywhen a superficial lesion is caused by the cancer, or an excision of theaffected portion when a more profound lesion is caused. Thepost-surgical rate of survival is 90%. The B stage is measured accordingto the degree of invasiveness of the cancer within the organs or thetissues surrounding the tumor. The treatment for these cases is usuallythe excision of the tumor and considering using chemotherapy and/orradiotherapy. The survival rate figures between 70-80%. The C stageinvolves the invasion of lymphatic nodules and the formation ofmetastases in the major blood vessels. The treatments include excisionof the diseased portions, chemotherapy with the combination of anadjuvant. The last stage, the D stage, distal metastases are present.Treatments include ablation of different isolated metastases (liver,lung, ovaries), as well as chemotherapy and/or palliative chemotherapy.The survival rate following this type of operation is usually less than5 years.

5.1.2 Chemotherapy

The most common drug used for chemotherapy is of 5-fluoro-uracil (5FU)it is administrated in intravenous form. Studies have demonstrated thatthe use of 5FU, after the excision of the tumor, is more beneficial forpatients in Duke's stage C than excision alone. With the desire toobtain the best results to vanquish colon cancer, combined treatmentsare prescribed to different patients in stages B or C. Many studies showthat with the combination of 5FU with an adjuvant, namely levamisole orleucovorin, better results are obtained for Duke's stage C. Levamisoleis known to enhance the efficacy of 5FU; with this combination, it isshown that there is a reduction in the recurrence of the cancer, andthat the mechanism is probably related to macrophage activation whichdestroys remaining tumor cells since it seems to positively regulate theimmune system. Leucovorin is a folic acid which is administered to avoidthe negative hematological effects and therefore to help keep thehealthy cells alive and to leave the cancerous cells susceptible to thecytotoxic action of 5FU. In general, studies agree that the treatmentsusing a combination with an adjuvant has a positive effect on thesurvival rate and on the recurrence time of the tumor after the ablationof the cancer when compared to the use of 5FU alone. 5FU acts by bindingto an enzyme within the cell allowing thymine synthesis during DNAreplication, and inhibiting it. Consequently, the cell, unable todivide, will die. Other drugs may act against colon cancer and arecurrently under clinical studies, or will be shortly. The differentdrugs are the following: Irinotecan (Campotasar, CPT-11), Oxaliplatine,Ralitrexed and Xeloda (Capecitabine). Irinotecan acts by inhibitingtopoisomerase I which is necessary to give a certain shape to the DNAduring transcription, translation and replication. Oxaliplatine acts onDNA by forming bridges in the DNA and thus inhibiting its synthesis andits replication. Ralitrexed has a role similar to 5FU since itinterferes in DNA synthesis by blocking the enzyme involved in thyminesynthesis. Xeloda is an oral pill which is transformed into 5FU afteringestion.

5.1.3 Mechanism of Cancer Cell Destruction by Chemotherapy

When the effect on the cells of a drug is known, it is possible tounderstand the mechanism leading the cell to become apoptotic. Being aninhibitor of thymine synthesis, 5FU causes DNA damage during cellulardivision by depriving one of the four pyrimidine bases constituting DNA.By this DNA damage, 5FU is responsible of activating p53, the proteinguardian of the genome. It is demonstrated that this protein, by itstranscriptional activity, can modulate the expression of the differentproteins involved in the process of apoptosis, for example Bax, Bak andBcl-2. Therefore, when the expression of Bax is increased and that theexpression of Bcl-2 is decreased, the chances that the mitochondrialmembrane potential is lost is increased, which sets in motion themitochondrial apoptotic process. These proteins are regulated by the p53protein, determines the sensitivity of the cell to chemotherapy becausein the majority of colon cancers, p53 is mutated. Despite the fact thatp53 is mutated, it is possible to observe the death of these cellsfollowing the addition of 5FU.

It has also been demonstrated that the treatment of cancerous cells with5FU induces Fas receptor expression and Fas ligand expression at thesurface of the cancerous cells. The induction of the expression of Fasreceptor thus allows a better chance of elimination of the cancerouscell by the immunonological cells. It has also been suggested that bythe induction of Fas receptor and ligand expression at the surface oftreated cancer cells, an autocrine, paracrine or fratricidal death mayfollow. Since the cells express both the receptor and the ligand, theremay be cross-linking between the receptor and the ligand of the samecell (autocrine), or the receptor on one cell and the ligand of anothercell (paracrine). Different teams of researchers have demonstrated thatchemotherapy-induced apoptosis involves the activation of the caspase-3and -8 regardless of whether the cell is of type I or type II. It issuggested that in the type I cells, two apoptosis initiation pathwaysare used during chemotherapy. Therefore, the treatment favors Fasreceptor aggregation, which causes the activation of the caspase-8,which in turn will directly activate the caspase-3, the caspase-8 mayalso activate the Bid protein by cleaving it and Bid will then activatethe mitochondrial apoptotic pathways. For type II cells, apoptosis iscontrolled by a mitochondrial pathway since the use of FADD inhibitorsdid not decrease chemotherapy-induced apoptosis this type of cells.Therefore, caspase-8 activation in type II cells occurs after thesignalling events in the mitochondrion.

Other studies have shown that DNA damage caused by chemotherapy or byirradiation increases the death receptor DR5 expression in ap53-dependant and independent manner.

We can observe the involvement of many proteins following a chemotherapytreatment. It is important to understand the mechanisms used for thistreatment since different cancers develop different tricks to avoiddeath.

6 Lactic Acid Bacteria

It is the scientist E. Metchnikoff (1845-1919) who proposed that thelongevity and the health of the Bulgarian people is attributable totheir ingestion of fermented milk products. It was well known thatcertain bacteria are pathogenic to the organism; thus, it was proposedthat these bacteria be substituted by yogurt bacteria since they hadlong been used without fear. Many characteristics exist in order todefine the good lactic acid bacteria: they must conserve their activityand their viability prior to consumption, they must survive thegastrointestinal tract, they must be able to survive and to proliferatein the intestines, and must eventually produce beneficial effects. Inaddition, the micro-organisms must not be pathological nor toxic.

Since then, many trials have been conducted in order to improve thestate of health by modification of the intestinal flora through livinglactic acid bacteria. Today, the beneficial effects of these lactic acidbacteria are well identified and there are attempts to explain themechanism(s) related to such benefits. Salminen's team has summarizedthe most important beneficial effects, supported by scientific evidencesuch as immunological modulation and reinforcement of the intestinalmucous barrier. Other teams have demonstrated that, in the mouse, cancergrowth and metastases may be inhibited by the Lactobacillus casei strainof bacteria. Different mechanisms are proposed in order to explain towhat such benefits would be due: the modification of the intestinalflora, adherence to the intestinal mucous membrane with the capacity ofpreventing the adherence of pathogenic bacteria or the activation ofpathogens, the modification of food proteins by intestinal microflora,the modification of bacterial enzymatic capacity, especially thosesuggested relating to the induction of cancer, and finally the influenceon the permeability of intestinal mucosa.

Most of the studies indicate a therapeutic potential of lactic acidbacteria and yogurt which is mainly due to the change ingrastro-intestinal micro-ecology. The efficiency of lactic acid bacteriais enhanced by their capacity of adherence to the intestinal wall sincethe adherent bacterial strains have a competitive advantage, importantto maintain their place in the gastrointestinal tract. On the otherhand, no bacterial strain has yet been shown to adhere in a permanentfashion. By increasing the quantity of lactic acid bacteria in theintestines, it is possible to eliminate growth of pathogenic bacteriawhich in turn will contribute to a reduction of infections. An intactintestinal epithelium with an optimal intestinal flora represents abarrier against invasions or colonisation by pathogenic micro-organisms,antigens and harmful compounds for the intestinal tract.

In general, the consumption of lactic acid bacteria acts by areinforcement of the non-specific immune response or acts as an adjuvantin the antigen-specific immune response. Studies on animals havedemonstrated that the lymphoid tissue associated to the intestines isstimulated by living lactic acid bacteria, resulting in a production ofcytokines and antibodies (IgA) and an increase of mitogenic activity ofthe cells forming Peyer plaques and splenocytes. In the studies on humancells, the production of cytokine, phagocytic activity, antibodyproduction, function of T cells and NK cell activity are increased bythe consumption of yogurt or when the cells are exposed to lactic acidbacteria in a in vitro.

Certain evidences indicate that the yogurt stimulating the immune systemmay be associated with the reduction of pathological incidences likecancer, gastrointestinal disorders and allergy symptoms.

6.1 Anti-Cancerous Properties

The lactic acid bacteria would have antineoplastic properties in avariety of cancer cellular strains of human and animal origin. In brief,the lactic acid bacteria reduce the viability of tumoral cells, reducecarcinogenesis induced in the colon and in the liver, inhibits mutagenicactivity and binds to potentially mutagenic compounds. Although nomechanism is known, it is suggested that the inactivation or theinhibition of cancer formation in the intestinal tract is induced.

There exists considerable interest for the metabolic activity of theintestinal microflora, especially in relation to colon cancer etiology.Studies have been conducted amongst others on the measure of keyenzymes: β-glucoronidase, azo-reductase, and nitro-reductase. Theseenzymes catalyze the conversion of indirect carcinogens into carcinogensin the intestines. By the absorption of lactic acid bacteria, thesewould reduce the activity of these key enzymes and would thus preventthe formation of tumors. An oral supplement of lactic acid bacteria (L.acidophilus) of human origin causes a significant reduction of thesethree key enzymes. These results have been partially confirmed by theMarteau team which recorded a reduction only in nitro-reductase in 9 ofthe subjects that had ingested lactic acid bacteria (L. acidophilus, B.bifidium) during 3 weeks. The studies have continued on an animal coloncancer model chemically induced by 1,2-dimethylhydrazine dihydrochloride(DMH). DMH activation occurs in the large intestine and it is thebacterial enzyme β-glucoronidase which transforms it into a potentialcarcinogen. The suppression of this enzyme can reduce the activation ofDMH and subsequently tumor formation. These studies show that theaddition of lactic acid bacteria can delay the formation of colon cancerby prolonging induction, indicating that the lactobacilli can slow tumordevelopment in the animal experimental model.

In summary, many conclusions are suggested related to the inhibitoryfunctions of lactic acid bacteria towards colon cancer. Besides, theincrease or the stimulation of immune functions could contribute inreducing the risk of the development or the reappearance of the cancer.Also, lactic acid bacteria could take the place of pathogenic bacteriawhich would be at the origin of the formation of mutagenic compoundscausing colon cancer.

There is therefore a need to find more efficient methods or methodshaving less secondary effects than the treatments already available fortreating the disease.

SUMMARY OF THE INVENTION

As mentioned here above, new properties of these bacteria have beendiscovered. Indeed, it has been discovered surprisingly that the effectof lactic acid bacteria, in particular those contained in the productsold under the trade name Bio-K+International, in combination with ananticancer agent can cause cellular apoptosis.

Many mechanisms can be at the origin of such phenomenon. For example,lactic acid bacteria can avoid the mutation that is at the origin ofcancers. It can also prevent the progression of tumors by reinforcingthe immune system.

The Applicant has discovered surprisingly that the use of lactic acidbacteria would have as an effect to prevent cancer formation, inparticular, colon cancer.

The Applicant has also discovered surprisingly that the use of lacticacid bacteria in combination with an anticancer agent has the effect ofincreasing the susceptibility of cancerous cells to apoptosis.

Thus, the present invention concerns the use of at least one lactic acidbacteria strain to reinforce an immune response in a mammal in order toprevent or to treat a cancer.

Another object is the use of at least one lactic acid bacteria strain tofacilitate the induction of apoptosis in cancer cells.

Another object is the use of at least one lactic acid bacteria strainfor the manufacture of a drug destined for the treatment or theprevention of cancer.

According to a preferred embodiment, the bacterial strain is in a liveor irradiated form. More particularly, this bacterial strain is of thegenus Lactobacillus and more preferably of the species Lactobacillusacidophilus such as the strain I-1492 filed at the CNCM and/orLactobacillus casei.

Another object of the present invention is to provide a composition totreat or to prevent a cancer, such as colon cancer. The composition ofthe present invention comprises an efficient quantity of at least onelactic acid bacterial strain such as previously and an acceptablepharmaceutical carrier. According to a preferred embodiment of theinvention, the composition also comprises an anticancer agent, such as5-fluorouracil.

According to another object, the present invention proposes a method toprevent or to treat a cancer in a mammal, characterized in that itcomprises the administration in said mammal of a composition such aspreviously defined.

Another object of the present invention concerns the use of lactic acidbacteria to increase apoptosis of cancerous cell.

Another object of the present invention concerns the use in combinationof lactic acid bacteria and an anticancer agent, such as 5FU for thetreatment of a colon cancer.

Another object of the invention is to provide a kit to prevent or totreat a cancer in a mammal, characterized in that it includes acontainer containing a composition such as the one previously defined.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a typical analysis of the percentage ofapoptosis by flow cytometry.

FIG. 2 is a diagram illustrating a model of the apoptotic process.

FIG. 3 is a diagram illustrating the hypotheses of release by the Bcl-2protein family.

FIG. 4 is a diagram illustrating the proteolytic maturation ofpro-caspase-3.

FIG. 5 is a diagram illustrating the recapitulation of the effects ofp53 activation.

FIG. 6 is a diagram illustrating the structure of membranous deathreceptor members and their interaction with the main cytoplasmiceffectors involved in apoptotic pathways.

FIG. 7 is a diagram illustrating the interconnections between twodifferent apoptotic transduction pathways.

FIG. 8 is a graph showing the optimal dose of 5-fluorouracil to obtain50% of cellular death.

FIG. 9 shows a visual presentation of diagrams of apoptosis obtained byflow cytometry.

FIG. 10 is a graph showing the effect of the composition according to apreferred embodiment of the invention on LS 513 cell viability.

FIG. 11 is a graph showing the effect of the composition according to apreferred embodiment of the invention on LS 513 cell apoptosis.

FIGS. 12 a, 12 b, 12 c, and 12 d are diagrams illustrating the measurefor apoptosis by flow cytometry. FIG. 12 a illustrates the cells nothaving been subjected to any treatment. FIG. 12 b illustrates cells inthe presence of 5FU in a concentration of 100 μg/ml. FIG. 12 cillustrates cells in the presence of lactic acid bacteria at aconcentration of 10⁸. FIG. 12 d illustrates the combination of cells,lactic acid bacteria (10⁸) and of 5FU (100 μg/ml).

FIG. 13 shows a Western blot illustrating the activation of caspase-3 bythe cells in the presence of a composition according to a preferredembodiment of the invention.

FIG. 14 is a graph showing a measure of apoptosis of LS 513 cells in thepresence of compositions according to a preferred embodiment of theinvention.

FIG. 15 is a graph showing the effect of the viability of LS 513 cellsby MTT.

FIG. 16 is a graph showing the effect of live lactic acid bacteriaversus heated lactic acid bacteria on LS 513 cell apoptosis.

FIG. 17 shows Western blots showing the effect of living lactic acidbacteria versus heated lactic acid bacteria on the activation ofcaspase-3.

FIG. 18 is a graph showing the measure of inherent apoptosis in lacticacid bacterial strains of the present invention.

FIG. 19 is a graph showing the apoptotic effect of the mixture of livingbacteria and of heated bacteria.

FIG. 20 is a graph showing the effect of the addition of butyric acidand of 5FU on LS 513 cell apoptosis.

FIG. 21 is a graph showing the effect of the composition according to apreferred embodiment of the invention on Fas receptor expression.

FIG. 22 is a graph showing the effect of the composition according to apreferred embodiment of the invention on Fas ligand expression.

FIG. 23 shows Western blots illustrating the effect of the compositionaccording to a preferred embodiment of the invention on the expressionof a protein involved in apoptosis, protein p53.

FIG. 24 shows Western blots illustrating the effect of the compositionaccording to a preferred embodiment of the invention on the expressionof a protein involved in apoptosis, protein p21.

FIG. 25 is a graph showing the effect of PKC on apoptosis.

FIG. 26 is a graph showing the effect of the inhibition of PKC onapoptosis.

FIG. 27 is a graph illustrating the effect of supernatant of the lacticacid bacteria of the invention on the induction of caspase-3 activity inLS 513 cells.

FIG. 28 shows photographs illustrating the effect the supernatant of thelactic acid bacteria of the invention on the coloration in fluorescenceof the nucleus of LS 513 cells.

FIG. 29 is a graph illustrating the effect of the supernatant of thelactic acid bacteria of the invention on the viability of LS 513 cells.

FIG. 30 is a graph illustrating the effect of the supernatant of thelactic acid bacteria of the inventions on the cellular gel of LS 513cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention therefore concerns the highlighting of the use ofnew properties of lactic acid bacteria strains in the prevention or thetreatment of a cancer. More particularly, the use of these lactic acidbacteria aims to facilitate the induction of cellular apoptosis of acancer.

The invention also concerns the implication of said lactic acidbacterial strains in methods and useful compositions in the treatment orthe prevention of a cancer, such as colon cancer.

According to a first embodiment, the present invention aims at the useof lactic acid bacterial strains to strengthen the immune response in amammal in order to prevent or treat a cancer.

According to a second embodiment, the present invention aims for the useof lactic acid bacteria to facilitate the induction of apoptosis incancer cells. By “facilitate the induction of apoptosis”, we mean aprocess by which the presence of lactic acid bacterial strains of theinvention positively modulates cellular death of a tumor, and preferablya tumor of the colon.

By “mammal”, we mean any living organism which may be subject to acancer, and this includes vertebrate beings such as in particular humanbeings and domestic and wild animals.

By “treat”, we mean a process by which the symptoms of cancer, andparticularly that of the colon, are reduced or completely eliminated.

By “prevent”, we mean a process by which the cancer, and particularlythat of the colon, is controlled or delayed.

According to a preferred embodiment of the invention, the bacterialstrain is of the genus Lactobacillus and preferentially in a living orirradiated form. More particularly, the bacterial strain is of thespecies Lactobacillus acidophilus or Lactobacillus casei. In the casewhere the species chosen is Lactobacillus acidophilus, the inventorsprefer advantageously to use the I-1492 strain deposited at the CNCM.

According to a third embodiment, the present invention in regarding theuse of these lactic acid bacterial strains for the preparation ofcompositions useful in the treatment or the prevention of cancer, suchas colon cancer. A composition according to the present inventioncomprises an efficient quantity of at least one lactic acid bacterialstrain and one acceptable pharmaceutical vehicle. Preferably, thecomposition of the invention includes a mixture of L. acidophilus and L.casei strains.

By “pharmaceutically acceptable”, we understand a vehicle that can beadministered without risk to a mammal, in particular to a human being,and this with little or no negative or toxic secondary effects. Such avehicle can be used for different functions. For example, it can be usedas a preservation, solubilizing, stabilizing, emulsifying, softening,coloring, odoring agent, or as an antioxydant agent. These types ofvehicles may be easily prepared by using methods well known by a personin the art.

According to a preferred embodiment, the composition of the presentinvention comprises, besides, an anticancer agent. To this effect, anyanticancer agent that could be useful in the present context is includedwithin the scope of the present invention However, 5-fluorouracil isadvantageously used as an anticancer agent. The composition of thepresent invention can also be part of a more complex therapeuticformulation which is useful in the treatment and the prevention ofcancer.

According to a fourth embodiment, the invention proposes a method toprevent or to treat a cancer, in particular a colon cancer, in a mammal.According to another closely related embodiment, the present inventionproposes a method to facilitate apoptosis of cells of a cancer in amammal, such as a human. These methods include, amongst others, thestage of administering to said mammal a composition according to thepresent invention.

The quantity or the concentration of lactic acid bacteria that isadministered to a human or to an animal or that is present in thecomposition of the invention is a therapeutically effective quantity. Atherapeutically effective quantity of lactic acid bacteria is thequantity necessary to obtain positive results without causingexcessively negative secondary effects in the host to which the lacticacid bacteria or the composition is administered. Moreover, an efficientquantity of lactic acid bacteria to treat a particular cancer is aquantity which is sufficient to attenuate or to reduce in any fashionthe symptoms linked to cancer. Such a quantity can be administered in asingle dose or can be administered according to a regime, by which it isefficient. The quantity of lactic acid bacteria according to the presentinvention can treat the cancer but, typically, it is administered inorder to attenuate the symptoms of cancer. The exact quantity of lacticacid bacteria or each of the components of the composition to beadministered will vary according to factors such as the type of cancerto be treated, the other ingredients in the composition, the method ofadministration, the age and the weight of the mammal, etc. . . .

The compositions according to the present invention can be presented inany solid or liquid form that is usual to pharmaceutical administration,that is for example for liquid administration forms, in a gel, or anyother support known by a person skilled in the art. Among thecompositions that are used, we can in particular cite compositions thatcan be administered orally. In the present case, the composition of theinvention can be administered in the form of food or food supplements.We can also cite injectable compositions more specifically destined forinjections in the blood circulation of humans.

A person versed in the art will know how to prepare compositions thatare pharmaceutically acceptable and determine, in function of manyfactors, the privileged method of administration and the quantity thatshould be administered. Among the factors that can influence his choiceswe find: the nature of the treatment, the exact nature of theingredients, active or not, entering in the composition; the stage ofthe disease; the condition, the age and the weight of the patient, etc.

The present invention also includes useful pharmaceutical kits, forexample, for the prevention or the treatment of a cancer, such as coloncancer. The kits comprise one or many containers further containing acomposition according to the present invention. Such kits can alsoinclude, if desired, one or many conventional pharmaceutical componentslike, for example, containers containing one or many pharmaceuticallyacceptable vehicles, or any other additional component, which will beobvious to a person skilled in the art. A kit according to the presentinvention can advantageously include instructions in pamphlet form or onany other printed support, indicating the quantities of the componentsto be administered, the instructions for administration, and/or theinstructions to mix the components.

The example here after will allow to highlight other characteristics andadvantages of the present invention.

EXAMPLE

The following example serves to illustrate the scope of the use of thepresent invention and not to limit its scope. Modifications andvariations may be made to it without going away from the spirit nor thescope of the invention. Eventhough one may use other methods or productsequivalent to those that we find hereinafter to test or to carry out thepresent invention, the material and the preferred methods are described.

Introduction

In the context of the present invention, in order to determine how thelactic acid bacteria help in the apoptosis of cancer, trials have beenconducted on the human colon cancer cell line LS-513. The lactic acidbacteria used constitute a mixture of Lactobacillus acidophilus andLactibacillus casei. The anticancer agent is 5 fluorouracil (5FU). Thiscompound acts as an inhibitor of the enzyme that synthesizes thymine.

Material and Methods

1 Lactic Acid Bacteria

1.1 Origin

The mixture of bacteria used for the different experiments is suppliedby the company Bio-K (Laval, P.Q. Canada). The mixture comprises acombination of Lactobacillus acidophilus I-1492, which is the subject ofthe international application no. PCT/CA97/00915, and of Lactobacilluscasei.

1.2 Preparation

The bacteria received in 9 mL of MRS complex medium (Difco Laboratories,Detroit, USA) are immediately multiplied in 100 mL of the same medium bytaking 100 μL of the bacterial suspension. After an incubation of 18hours in an incubator at 37° C., 10 mL of glycerol is added to the 100mL mixture which is then divided into 1 mL aliquots in many sterileplastic flasks that can contain 1.5 mL. These flasks are stored in afreezer at −80° C.

For the different stimulation protocols, a flask is thawed and put into9 mL of MRS and incubated for 18 hours at 37° C. After this incubationperiod, a subculture is done. A volume of 100 μL is taken and added to 9mL of MRS which is also incubated for 18 hours at 37° C. After theseincubations, the bacteria are washed twice in sterile PBS and collectedby centrifugation at 3500 RPM during 10 minutes. They are then suspendedin a final volume of 9 mL of sterile RPMI containing 10% foetal bovineserum and are then ready to be used for a cellular stimulation. Duringthe different experiments, the bacteria are used in a heated, irradiatedand live form.

1.3 Heating

The bacteria are heated for 40 seconds at “high” in a microwave oven(General Electric, Turntable Microwave Oven, 700 Watt) in order toobtain a 100° C. temperature and to produce the mixture of bacteriawhich will be used as heated bacteria. This step occurs in a closedglass container.

1.4 Irradiation

In order to produce the mixture of irradiated bacteria, the tubes oflive bacteria are irradiated at a minimal dose to obtain a 100%mortality, i.e. 5kGy. The mixtures are irradiated in a Gammacell-220(MDS, Nordion, Laval, P.Q., Canada) using Cobalt-60 (⁶⁰Co) as a gammaray emitting source.

In order to do a bacterial count to obtain a concentration of bacteriaper 1 mL of this suspension is added to 9 mL of peptoned water, anisotonic solution containing 0.1% of bactopeptone (Difco Laboratories,Detroit, USA). Serial dilutions are made. Then, 1 mL of these dilutionsis taken and put in a Petri dish to which one adds 10 mL of MRS to 1.5%agar (Difco Laboratories, Detroit, USA) to allow a counting after a 48hour incubation in an incubator at 37° C. Each sample is done induplicate.

2 Cancer Cells

2.1 Origin

The LS 513 cell line (ATCC, Rockville, Md., USA) is a continuous coloncancer line of human origin.

2.2 Culture

The cell line being adherent, one must detach the cells with the help ofa trypsin-EDTA solution (Gibco, Burlington, ON, Canada) in order toresuspend them in RPMI supplemented with L-glutamine and 10% FBS whichwe can thus call “complete RPMI”. The plates are done the day before thestimulation to allow the cells to adhere to the plates. The day of thestimulation, the different products are added at the desiredconcentrations and then the cells are incubated during a determinedperiod for each type of experiment, in an incubator at 37° C., 5% CO₂and saturated in humidity.

3 Co-Cultures of Cancer Cells and Bacteria

3.1 Addition of Bacteria

Once the culture plates are ready to be used, that is when the cellshave had time to adhere, and the bacteria ready to be used, the latteradded according to the stimulation protocol in wells containing theadherent cells and the “complete RPMI” medium. An incubation is doneduring a given time based on the experiment to conduct.

3.2 Experiment Involving the Addition of Butyrate

The plate of cancer cells containing 3×10⁵ cells per well is incubatedovernight to allow the cells to adhere. Then, the differentconcentrations of butyric acid (Sigma, St-Louis, USA) are added to thewells containing the adherent cells and the culture medium, the completeRPMI. After this addition, a 48 hour incubation is done in order tomeasure the percentage of apoptosis according to the technique describedhereinafter.

3.3 Collecting and Studies of Supernatants

The goal of this experiment is to verify if the presence of bacteriamodifies the pharmacological presentation of 5FU. The first stimulationis made on adherent cells for a period of 48 hours in the presence ofthe different stimulation products. Following this incubation, thesupernatants are collected and added onto a new culture of freshlyadherent cells. Then, a second incubation of 48 hours is done then thecells are harvested and the percentage of apoptosis is measured.

4 Measures of the Vialibity of Cancer Cells

4.1 Proliferation

4.1.1 MTT

The cells are prepared as described above. The test is done in a 96-wellplate. A volume of 100 μL of a concentration of 3.3×10⁵ cells per mL isdeposited in each well, except in the first row which will serve as a“blank”. Once the stimulation products are added, an incubation of 48hours is made. Following the incubation, the supernatants are removed byaspiration, and the cellular sheet is washed, by delicately adding 200μL of PBS in each well, and by rapidly decanting the added PBS in thesink. Then, a solution of MTT (Sigma, St-Louis, USA) 5× diluted incomplete RPMI is added to the wells and an incubation of 5 hours at 37°C. is done. Then, the solution is decanted and another solution is addedwith the goal of dissolving the crystals formed in the live cells. Thissolution is composed of 50% of dimethyl formamide and 12% sodium dodecylsulfate (SDS). An incubation of 18 hours is required to dissolve all thecrystals. Following this incubation, the plate is read in aspectrophotometer (Mandel Scientific Company, Bio-Tek Instruments,Microplate EL309 Autoreader) at 540 nM. Each sample is made intriplicate, and the obtained values are averaged. The average value ofthe standard is the “100%” of living cells. In order to obtain thepercentage of other samples, it suffices to do a cross product.

4.2 Apoptosis

4.2.1 Flow Cytometry

A concentration of 5×10⁴ cells per mL, with 6 mL per well, is used inorder to obtain 3×10⁵ cells per sample. The cells are prepared asindicated above. The different products are added to the wells, and anincubation of 48 hours is done.

Following the incubation, the supernatants of the cells are collected indifferent 15 mL tubes and centrifuged at 1500 RPM during 5 minutes. Thecellular pellets are then collected by decantation of the supernatantand put on ice. This stage consists in recuperating the cells insuspension. Afterwards, the adherent cellular sheet is washed with 0.5mL trypsin-EDTA, then 0.2 mL trypsin-EDTA is added to each well to allowthe cells to detach when they are incubated for around 8 minutes in theincubator at 37° C. The cells are suspended in 3 mL of complete RPMI 10%FCS to stop the enzymatic activity of trypsin. The cellular suspensionis centrifuged at 1500 RPM for 5 minutes in the tubes used for flowcytometry. Afterwards, the two cellular pellets (cells in suspension andadherent cells) are combined and washed twice with cold PBS supplementedwith 0.25% EDTA in order to avoid the formation of agglomerates ofcells. Following the washings, 0.5 mL of propidium iodide solution isadded to the cellular pellet. The solution is composed of 0.1% sodiumcitrate (Fisher Scientific, New Jersey, USA), 0.1% de TritonX-100(Sigma, St-Louis, USA), 50 μg/mL of RNase (Sigma, St-Louis, USA) and of20 μg/mL of propidium iodide (Sigma, St-Louis, USA). A 15 minuteincubation at 4° C. is done to then analyse the samples by flowcytometry (Coulter Epics XL-MCL). FIG. 1 summarizes a typical resultfrom a flow cytometry analysis. Different peaks are formed due to thedifferences in fluorescence existing between each phase of the cellularcycle. The more the content of intact DNA is high, the more thefluorescence is high and vice-versa. The program measures the percentageof fluorescence that the large spike forms under the corresponding spikeof G0-G1, this spike represents pieces of fragmented DNA, consequence ofthe cleavage of DNA by different enzymes activated during apoptosis.

5 Measure of the Expression of the Proteins Involved in Apoptosis (p53,p21, Caspase 3, Bax)

5.1 Western Blotting

5.1.1 Protein Stimulation and Extraction

A total of around 6×10⁵ cells per sample are used. The following day,when the cells have become adherent, the stimulation products are added.The different stimulation products are 5-fluorouracil (100 μg/mL) andthe bacteria, live or heated, at a concentration of 1×10⁸ bacteria parmL. These products will bring the cells to become apoptotic by themodulation of different proteins involved in the process. It is thismodulation, increase in expression or protein activation, that thetechnique allows to verify. Following different stimulation times (sinceit concerns kinetics), the cells are collected and centrifuged at 1500RPM for 5 minutes. Then, 50 μL of lysis buffer composed of 50 mMTris-HCl pH 7.5, NaCl 150 mM, Nonidet P-40 1% (Roche Diagnostics, Laval,P.Q.) as well as a Complète™ pill (containing protease inhibitors)(Roche Diagnostics, Laval, P.Q.), are added to the cellular pellet whichis then incubated 30 minutes on ice. The volume of 50 μL is collectedand put into a 1.5 mL micro-tube. A centrifugation of 10 minutes at 15000 RPM is done in order to precipitate the cellular debris. Thesupernatant is collected and an equal volume of “sample buffer” isadded. The “sample buffer” is composed of 100 mM Tris-HCl pH6.8, 2% SDS,20% glycerol and of 0.006% of bromophenol blue. At the end, the samplesare aliquoted by volume of 20 μL and are stored at a temperature of −20°C.

5.1.2 Protein Separation and Identification

The samples of protein are separated on a polyacrylamide-SDS gel at 4%and 12% using the “mini-PROTEAN” machine from Bio-Rad. The proteinsmigrate within an electrical current of 200 volts during 45 minutes. Themigration occurs in an “electrode buffer” at pH 8.3 composed of 1.5%Tris-Base, of 7.2% glycine and of 0.5% SDS in mili-Q water. Afterwards,the proteins are transferred on a “Hybond ECL” nitrocellulose membrane(Amersham Pharmacia Biotech Inc., Baie d'Urfé, Quebec) with the help ofthe Bio-Rad transfer machine during one hour at 100 volts in a transferbuffer composed of 0.58% Tris-Base, of 0.29% glycine, of 0.037% SDS andof 20% methanol. Following the transfer, the membrane is “blocked” witha blocking solution composed of 0.1% PBS-Tween 80 at 0.1%, as well as 5%powdered skimmed milk for one hour at room temperature with agitation.Then, the first labelling is done with the antibody recognizing thetargeted protein. The antibody is diluted in the blocking solutionaccording to a concentration given by the provider. After one hour oflabelling, a 15 min. washing is done followed by two washings of 5minutes with the blocking solution. The second labelling is done with asecond antibody which recognizes the first antibody and which is coupledto the peroxidase. A one hour incubation is done with this secondantibody which is also diluted in the blocking solution at aconcentration given by the provider. As soon as this incubation isfinished, a 15 minute washing, as well as two 5 minute washings, aredone with a solution of 0.1% PBS-Tween 80 without powdered skimmed milk.In order to detect the different proteins, an ECL solution (AmershamPharmacia Biotech Inc, Baie d'Urfé, P.Q., Canada), which leads to theactivation of the peroxidase, is added to the membrane according to theinstructions of the provider. Afterwards, the labellings are revealed onphotographic paper (hyperfilm ECL, Amersham Pharmacia Biotech Inc., Baied'Urfé, P.Q., Canada) which will be labelled by the activatedperoxidase. The photographic paper is then developed. Table 1 summarizesthe proteins targeted and the reactants used.

TABLE 1 The antibodies used for the different proteins to be identified.Protein Description Dilution Company Primary Antibody p53 Purified human  1:1000 BD PharMingen, anti-p53 Mississauga, Ontario Iso type: mouseIgG_(2a) p21 Purified mouse   2:1000 BD PharMingen, anti-p21Mississauga, Ontario Iso type: mouse IgG₁ Caspase 3 Anti-caspase 3  1:1000 BD PharMingen, rabbit polyclonal Mississauga, Ontario BaxAnti-Bax   1:1000 Santa Cruz California, USA Secondary Antibody IgGMouse anti-IgG   3:10000 Sigma, St-Louis, USA Coupled to peroxidaseDeveloped in goat IgG Rabbit anti-IgG 2.5:10000 Sigma, St-Louis, USACoupled to peroxidase Developed in goat6 Measure of the Expression of Markers on the Cellular Membrane (Fas,Fas L)6.1 Flow Cytometry

A total of about 5×10⁵ cells is used per sample. The cells are preparedas mentioned previously. A 24 hour incubation is done after the additionof different stimulation products (5FU, live or heated bacteria andothers according to the experiments that are conducted).

Following the incubation, the cellular sheets are washed with 0.5 mL oftrypsin-EDTA, then 0.2 mL trypsin-EDTA is added to the cellular sheets,which are then placed in the incubator at 37° C. for 10 minutes. Oncethe cells are detached, 3 mL of complete RPMI are added, and thecellular suspension is then centrifuged during 5 minutes at 1500 RPM.The supernatants are decanted and the cells are placed on ice. Then, 20μL of the antibody solution against the Fas receptor (BD PharMingen,Mississauga, Ontario) or Fas ligand (BD PharMingen, Mississauge,Ontario) are added to the cellular pellet to which 50 μL of “flowcytometry buffer” composed of 1×PBS, 1% BSA, 0.02% sodium azide and0.25% EDTA were previously added. An incubation of half an hour on icein the dark is required. Then, two washings with 4 mL of “flow cytometrybuffer” are done, by centrifugation at 1,500 RPM during 5 minutes. Tothe cells labelled with the Fas ligand antibody, a quantity of 0.25 μLper tube of streptavidine-PE (BD PharMingen, Mississauga, Ontario) isadded, following the first incubation and the two washings. A secondincubation is done for 30 minutes in the dark, followed by two otherwashes. At the end of a labelling, 250 μL of paraformaldehyde solution(1×PBS, 2% paraformaldehyde) and 250 μL of “flow cytometry buffer” areadded in order to fix the labellings. The tubes are wrapped in aluminiumpaper and placed at 4° C. until the analysis of the samples.

7 Measure of the Expression of the Nurr77 Gene

7.1 RNA Extraction

3×10⁵ cells are used per sample. A 3 hour incubation is done followingthe addition of the different stimulation products. The cells are thencollected with the help of a scraper, and centrifuged at 1500 RPM for 5minutes. The total RNA of each sample is removed and purified by usingthe High Pure RNA kit from Roche Diagnostics (Laval, P.Q., Canada) asinstructed by the manufacturer. The concentration of RNA is thenmeasured with the machine (Pharmacia Biotech, Gene Quant RNA/DNACalculator), then adjusted to 92 ηg/μL.

7.2 RT-PCR

The LightCycler RNA amplification SYBR Green 1 kit from RocheDiagnostics (Laval, P.Q., Canada) is used in order to accomplish thereverse transcription reaction (RT) and the polymerase chain reaction(PCR). The LightCycler principle is very similar to that of theThermocycler. The major difference consists in the possibility, with theLightCycler, of observing the amplification at each cycle, thanks to afluorescent molecule called SYBR Green 1 which inserts itself into eachdouble strand formed. The more double strands formed, the morefluorescence is observed with the help of the program included with theLightCycler. The two reactions, RT-PCR, are done in a capillaryspecially constructed for the LightCycler (Roche, Laval, P.Q., Canada)and it is sufficient to make a single mixture of the products containedin the kit and to use this ensemble according to the instructions of themanufacturer.

Before doing an amplification, it is necessary to finalize certainconditions. For example, the concentration of MgCl₂, the temperaturesand the time. An ideal concentration of MgCl₂ is to be determined. Forthe present amplification, a concentration of 7 mM proved to be thebest. The concentration of the primers is 0.5 mM, as suggested by themanufacturer. In the case of the Nurr77 gene, the primer sequence usedfor the positive strand is 5′-CGACCCCCTGACCCCTGAGTT-3′ (SEQ. ID. NO: 1)and the one for the negative strand is 5′-GCCCTCAAGGTGTTGGAGAAGT-5′(SEQ. ID. NO: 2) (Kang, J-H, Biol. Pharm. Bull.) The amplification bythese primers gives 658 base-pairs. The programming of the LightCyclermachine is described in the instruction manual provided by themanufacturer. Two other parameters vary in the amplification program:amongst others in the hybridization segment, the temperature it usesvaries according to the primers. The temperature (fixed at 5° C. lessthan the hybridization temperature of the primers (Tm) is calculated bythe following formula: Tm=2° C. (A+T)+4° C. (C+G). For the primers used,the Tm is 64° C. The second parameter is the incubation time for theelongation, always found in the amplification program; it is determinedby the following formula t=(number of base pairs from the amplifiedproduct÷25) seconds. In our case, the number of bases being 658, we thusobtain 26 seconds. Following the 35 cycles necessary to amplify thewanted part of the gene, a point of fusion curve is made, using atemperature of 10° C. above the hybridization temperature. Therefore,the amplifications are subject to a progressive increase of thetemperature and at each degree the fluorescence is measures andregistered. By increasing the temperature in a progressive fashion, thedouble strands formed detach when the temperature becomes high enough,and it is thus at this temperature that a decrease in fluorescence isobserved. The program makes a fusion point curve with the registeredfluorescences and, afterwards, it measures the derivative of thefluorescence in function of the temperature. By knowing the theoreticalfusion temperature of the amplification product, it is possible toobtain the value of the area below the curve of the spike peak formed bythe separation of the strands of the amplification product at thistemperature. Since the machine does not allow to visualise the number ofamplified base pairs, a migration on a 2% agarose gel with a base pairmarker allows the verification following staining with ethium bromidecoloration at a concentration of 0.5 μg/mL for 15 minutes.

8 Treatments with PKC Inhibitors or Stimulators

The stimulations made with the PKC inhibitors and stimulators areaccomplished in the same way the stimulations by bacteria and by 5FU.First of all, the cells are prepared the day before to allow them toadhere, and the day of the stimulation, the different stimulationproducts are added at the same time as the GÖ 6976 (Sigma) inhibitorand/or the PKC stimulators, ionomycine (Sigma) and PMA (phorbol12-myristate 13-acetate) (Sigma). Then, a 48 hour incubation is done,the cells are collected and the percentage of apoptosis is measured.

9 Cytokine Dosage

9.1 TNF Dosage by Bioassay

It is possible to measure out the quantity of TNF in a supernatant withthe help of the L929 cell line which is a mouse fibroblast linesensitive to the cytotoxic action of TNF. The principle of this bioassayis simple: the more TNF there is in the supernatant added to the L929cellular sheet, the more cellular death will occur. We can then measurethe rate of the remaining live cells. First, the cells are cultured incomplete RPMI 1640+5% FCS. The cells detach themselves from the flaskwith the help of trypsin-EDTA (Gibco, Burlington, ON, Canada) byincubating around 1 minute at 37° C. A cellular count is done to preparea suspension of 3.3×10⁵ cells/mL for the bioassay.

A volume of 75 μL is deposited in each well of a 96-well plate. All thelines receive this volume except the first which is used as an emptycontrol. Following a 24 hour incubation, a volume of 25 μL ofactinomycin D at a 2 μg/mL concentration is added to all the wells ofall the lines, except the second line which serves as a control ofactinomycin D and this in order to stop cellular growth. Then, thedifferent samples are added starting from the fourth line with 100 μLper well, and this in triplicate. The third line stays empty since itwill serve as a positive control, i.e. it will represent the maximumnumber of cells since there is no cytotoxic agent added. With the addedsamples, successive dilutions are made from the 100 μL that are dilutedin series in the 8 following rows. When the samples are diluted, theyare incubated from 16 to 20 hours at 37° C.+5% CO₂. After thisincubation, the supernatants are disposed of and the cells are fixed tothe bottom of the well with the help of a 5% formaldehyde solution with100 μL per well for 5 minutes. Then, the plates are emptied and rinsed 3times with running water. The cells that have remained fixed are thendyed with crystal violet with 50 μL per well for 5 minutes. Afterwards,the plates are emptied and the excess dye is eliminated by 3 rinses withrunning water. Once the plates have dried well, 100 μL of 33% aceticacid solution is added to each well to dissolve the crystal violetabsorbed by the fixed cells. The absorbency is read at a wave length of540 nm in a plate spectrophotometer, using column 1 as a reference(“Blank”).

In order to calculate the number of TNF units, we consider a unit of TNFcorresponding to the inverse of the dilution factor giving 50%cytotoxicity. In order to calculate the percentage of cytotoxicity foreach sample, the following equation is used.

${\%\mspace{14mu}{cytoxicity}} = {\frac{O.D.\mspace{11mu}{sample}}{O.D.\mspace{11mu}{positive}} \times 100}$

Therefore, the O.D. of the sample is the average of the threeabsorbencies obtained for a dilution following the reading of theplates. The O.D. of the positive control is the average of the wells ofline 3. The % cytotoxicity is calculated for each dilution. Afterwards,a linear regression curve is plotted for the dilutions of a sample, thedilution factor (=x) and the % cytotoxicity (=y) and the 50% point isfound with the help of the equation for the curve. The inverse of thedilution (2^(x)) is equivalent to the number of units of TNF in theinitial non diluted sample. The results are expressed in U/mL.

Results

1) Search for the Optimal Dose of 5-Fluorouracil

FIG. 8 shows the measure of apoptosis by flow cytometry followingexposure to increasing doses of 5-fluorouracil (5FU) in order to obtainan ideal concentration yielding 50% mortality. A total of 3×10⁵ cells isput in the presence of different concentration of 5FU during 48 hours.Then, the DNA content of the cells is labelled with propidium iodide andanalysed by flow cytometry in order to obtain the percentage ofapoptosis. The following results are the average of two independentexperiments.

FIG. 9 illustrates a diagram representing one of the two experimentsconducted in order to obtain the ideal concentration of 5FU. The machinemeasures the number of events under the G1 peak in relation to thenumber of events of the sample which yields a percentage result. Sincewe know that the events under G1 corresponds to cleaved DNA, sign ofapoptosis, we thus attribute this percentage as the value of apoptosis.

2) Action of the Combination of 5-fluorouracil and Live Bacteria on theViability of LS 513 Cells

FIG. 10 illustrates the viability of colon cancer cells and dosed by MTTafter an incubation of 48 hours in the presence or absence of5-fluorouracil (5FU) (2.5 μg/mL) and of live bacteria (B) at differentconcentrations (10⁶-10⁸). A total of 3.3×10⁴ cells per well in a 96-wellplate is put into contact with the different stimulation products. Thecoloration produced by the MTT reaction with the live cells is evaluatedon a spectrophotometer at a wave length of 540 nm. Each sample is theaverage of 3 different wells.

FIG. 11 illustrates the effect of live bacteria and of 5FU on theapoptosis of LS 513. Live lactic acid bacteria (B) at differentconcentrations (10⁶-10⁸) and of 5-fluorouracil (5FU) (100 μg/mL) areadded to the LS 513 cells. The measure of apoptosis by flow cytometry isdone following an incubation of 48 hours. The labelling of DNA with apropidium iodide solution allows to observe the percentage of cellshaving cleaved DNA (under-G1) produced following incubation. The controlis cells without treatment.

FIG. 12 shows examples of flow cytometry diagrams to measure apoptosis.These 4 diagrams represent 4 different samples produced during anexperiment. The number of events under G1 gives a percentage in relationto the rest of the stages of the mitotic cycle. The control (A) beingthe cells without treatment, image B is composed of cells put in thepresence of 5FU at a concentration of 100 μg/mL, image C is that wherelive bacteria at a concentration of 10⁸ were combined to the cells, andthe last image (D) represents the combination of cells, of live bacteria(10⁸) and of 5FU (100 μg/mL).

FIG. 13 illustrates a Western blot of caspase 3 and this in itspro-active form. An incubation of 48 hours in the presence of livebacteria at a concentration of 10⁸ and of 5-fluorouracil (5FU) at aconcentration of 100 ng/mL was done. From this incubation, the proteinswere removed and migrated on a polyacrylamide gel. Afterwards, they weretransferred to a nitrocellulose membrane and labelled with a specificantibody for caspase 3 and this at a concentration of 1:1000.

3) Action of the State of the Bacteria

3.1) Live Bacteria versus Irradiated Bacteria

FIG. 14 shows the measure of LS 513 cellular apoptosis in presence oflive bacteria, irradiated bacteria and 5FU. This figure shows morespecifically the measure of apoptosis by flow cytometry by thepercentage under G1 with the help of a propidium iodide label (20μg/mL). The colon cancer cells are put in the presence or absence of5-fluorouracil (5FU) (100 μg/mL) and of living bacteria (B) or ofirradiated bacteria (C) at different concentrations (10⁶-10⁹) and thisfor a period of 48 hours. The controls are cells without treatment.

3.2) Live Bacteria versus Heated Bacteria

FIG. 15 shows the measure of the viability of LS 513 cells by MTT. Moreparticularly, this Figure shows the effect of live bacteria (B) and ofheated bacteria (C) at different concentrations (10eX) in presence orabsence of 5-fluorouracil (5FU) (A)(2.5 μg/mL) on the viability of LS513 after an incubation of 48 hours. These values are obtained byspectrophotometer reading (540 nm), by the coloration due to MTT whichstains the functional mitochondria thus only those of the living cells.The cellular concentration used is 3.3×10⁴ cells per well. The followingresults are the average of three wells of a 96-well plate.

FIG. 16 shows the effect of live bacteria versus the heated bacteria onthe apoptosis of LS 513 cells. The analysis of the percentage under G1by DNA labelling was obtained with propidium iodide. A total of 10 000events are treated per sample. An incubation of 48 hours in presence oflive bacteria (B) and of heated bacteria (C) and this at differentconcentrations with or without 5-fluorouracil (100 μg/ml) are thedifferent samples illustrated in the figure.

FIG. 17 shows the effect of live or heated bacteria on caspase 3activation.

4) Possible Mechanisms Inherent to Bacterial Cultures

FIG. 18 shows the measure of apoptosis inherent to strains of lacticacid bacteria of the invention. A first stimulation was made during 48hours. Certain wells did not contain any cells (F). The differentsupernatants (D) were collected and added on a fresh cellular culture(E). The concentration of 5-fluorouracil (5FU) is of 100 μg/mL and twodifferent concentrations are used for the living bacteria (B) and theheated bacteria (C) which are 1×10⁷ and 10⁸. The measure of apoptosis isdone by flow cytometry by DNA labelling with propidium iodide.

FIG. 19 shows the apoptotic effect of the mixture of live bacteria andheated bacteria. The measure of apoptosis by flow cytometry was takenafter an incubation of 48 hours. The LS 513 cell line is put in thepresence of a given concentration of 5FU (100 μg/mL) as well as in thepresence of live bacteria (B) and of heated bacteria (C) at twodifferent concentrations (107 and 108). The control is composed of cellswithout treatment.

FIG. 20 shows the effect of adding butyric acid and 5FU on LS 513cellular apoptosis. A dose of 5FU (100 μg/mL) as well as different doses(2 mM and 4 mM) of butyric acid (ba) are added to the cancerous coloncells. Apoptosis is measures after a 48 hour incubation.

5) Possible Mechanism Inherent to Tumor Cells

FIG. 21 shows the effect of the composition according to a preferredembodiment on Fas receptor expression.

FIG. 22 shows the effect of Fas ligand expression.

FIG. 23 illustrates the effect of the composition according to apreferred embodiment of the invention on p53 protein expression.

FIG. 24 illustrates the effect of the composition according to apreferred embodiment of the invention on p21 protein expression.

FIG. 25 illustrates the effect of PKC activation on apoptosis.

FIG. 26 illustrates the effect of PKC inhibition on apoptosis.

6) Characterization of Lactic Acid Bacteria Supernatant of the Inventionon the Apoptosis of LS 513 Intestinal Tumor Cells

The apoptotic potential of bacterial supernatants on the LS 513 line,tumoral line was analysed. The results obtained on the activity ofcaspase-3 demonstrate that the supernatants, even in the presence of5-fluorouracil (2.5 μg/mL and 100 μg/mL) do not activate this caspase ina significant way (FIG. 27). Instead, in the presence of 100 μg/mL of5FU, the supernatants inhibit the activity of caspase-3. The fluorescentcoloration of the nucleus of the cancer cells, with the help of the DAPItechnique, did not allow to bring out nuclei where the chromatin iscondensed, a fundamental characteristic of apoptotic cells. Thecoloration of nuclei is typical of healthy and live cells (FIG. 28,enlargement at 100× and 630×). In addition, the flow cytometry studieswith the help of propidium iodide do not demonstrate cellular death(FIG. 29) nor significant disruption of the phases of the cellular cycle(FIG. 30). All of these results indicate that the bacterial supernatantsdo not induce apoptosis in the LS 513 cells.

Discussion

The action of intact bacteria, that is that are live and irradiated, onthe cancer is the same. However, the non intact bacteria, for examplethe cells destroyed by the heat, seems to have an action contrary to theintact bacteria.

In addition, it has been noticed according to the work conducted in thecontext of the present invention that the efficacy of an anticanceragent such as 5FU is considerably increased in the presence of intactbacteria. This efficacy would be dose dependent. A more rapid apoptosisin the presence of live bacteria is noticed.

The presence of heated bacteria with 5FU would increase the expressionof p21 protein. There could thus be modulation via an unknownapoptosis/cellular cycle (p21) regulator protein receptor. An increaseof the p21 protein was noticed when there is less apoptosis and adecrease of the protein when there is more apoptosis.

Butyric acid is a product of live lactic acid bacteria and is present inthe intestine. This product causes apoptosis in colon cancer cells invitro. There will be a synergy between butyrate and 5FU on colon cancer.It has also been noticed that butyric acid inhibits the in vivo growthof human colon cancer on mice.

In view of what precedes, live lactic acid bacteria enter in synergywith 5FU to decrease the number of cancer cells in culture (MTT) or toincrease apoptosis in the latter (flow cytometry).

The irradiated lactic acid bacteria have the same action as livebacteria whereas heated bacteria would have the opposite action. Hence,the intact form of lactic acid bacteria is necessary for their actionagainst the tumoral cells. In addition, the action of bacteria is alsodependent and proportional to the dose. The property to induce or tomodulate apoptosis by the lactic acid bacteria of the invention wascorroborated by the experiment demonstrating the non-apoptotic effect ofthe supernatant of said bacteria.

The expression of the caspase 3 as well as that of the p21 protein ismodulated by live lactic acid bacteria in the same way as apoptosis.

Lactobacillus acidophilus (accession number I-1492) herein described wasdeposited on Nov. 15, 1994 at the Collection Nationale de Cultures deMicroorganismes (CNCM; an International Depositary Authority, whose fullpost office address is Institut Pasteur, 28 Rue du Docteur Roux,F-75724, Paris, CEDEX 15, France) according to the provisions of theBudapest Treaty.

1. A method to facilitate induction of apoptosis of a cancer cell in amammal having cancer, the method comprising administering to the mammalLactobacillus acidophilus I-1492 deposited at the CNCM.
 2. The method ofclaim 1, wherein said Lactobacillus acidophilus I-1492 is intact.
 3. Themethod of claim 1, further comprising administering to the mammal astrain of Lactobacillus casei.
 4. The method of any one of claims 1, 2or 3, further comprising administering an anticancer agent.
 5. Themethod of claim 4, wherein the anticancer agent is 5 fluoro-uracil. 6.The method of claim 1, wherein said cancer cell is a colon cancer cell.7. A method to facilitate apoptosis of cancer cells in a mammal havingcancer, wherein the method comprises administering to said mammal acomposition comprising a lactic acid bacteria strain and apharmaceutically acceptable vehicle, said strain being Lactobacillusacidophilus I-1492 deposited at the CNCM.
 8. The method according toclaim 7, wherein said mammal is a human being.
 9. The method of claim 7,wherein the bacterial strain is intact.
 10. The method of claim 7 or 9,wherein said composition further comprises a strain of Lactobacilluscasei.
 11. The method of claim 7 or 9, further comprising administeringan anticancer agent.
 12. The method of claim 11, wherein the anticanceragent is 5 fluoro-uracil.
 13. The method of claim 2 or 9, wherein saidintact Lactobacillus acidophilus I-1492 is in a live form.
 14. Themethod of claim 2 or 9, wherein said intact Lactobacillus acidophilusI-1492 is irradiated.
 15. A method to facilitate induction of apoptosisof a cancer cell, the method comprising contacting the cancer cell withLactobacillus acidophilus I-1492 deposited at the CNCM.